Over the past decade, our group has focused on application of the tools of molecular biology to the development of insulin secreting cell lines that might serve as islet surrogates for transplantation therapy of diabetes. The focus of this proposal is on the design and implementation of methods for preventing impairment of function and/or islet cell damage by inflammatory cytokines and other small molecular weight mediators of the immune response. This renewal application was first funded in 1998 as part of an RFA entitled "Cellular and Molecular Approaches to Achieving Euglycemia." In the ensuing three years, we have made several discoveries pertaining to methods for protecting insulin secreting cells from damage by the immune system. Most important among these has been the development of a method for selecting cells that are resistant to the combined cytotoxic effects of the inflammatory cytokines IL-1 beta + IFN-gamma, involving culture of INS-1 insulinoma cells in iteratively increasing concentrations of these agents and collection of surviving cells. We have also demonstrated that resistance to IL-1 beta is related to impairment of NF-kB translocation and inducible nitric oxide synthase (iNOS) induction, while the resistance to IFN-gamma is due to a large induction in expression of signal transducer and activator of transcription (STAT)-1 alpha. Moreover, we have demonstrated that adenovirus-mediated overexpression of STAT-1 alpha confers resistance to IFN-gamma and IFN-gamma-IL-1 beta-induced cytotoxicity. Finally, we have recently learned that our selection strategy also confers resistance to cytokine-induced impairment of insulin secretion. In our view, these studies provide the platform from which novel and robust genetic engineering strategies can be developed for protecting insulin secreting cells from the immune system. To this end, the specific aims of this grant are: 1) To investigate the mechanisms by which resistance to IL-1 beta-induced cytotoxicity is conferred in cells subjected to our selection protocol; 2) To investigate the mechanism by which STAT-lalpha overexpression in insulinoma cells blocks cytokine-induced cytotoxicity; 3) To investigate the mechanism by which cytokines cause impairment of glucose-stimulated insulin secretion, and to determine the mechanism by which our newly developed selection and engineering methods confer resistance to this impairment. These studies have the potential to foster new methods for preserving beta-cell mass in both major forms of diabetes.